Mutagenesis, biochemical characterization and X-ray structural analysis of point mutants of bovine chymosin.
Chymosin B point mutants, A115T and G243D (chymosin A), were expressed in Escherichia coli and Trichoderma reesei respectively, characterized biochemically, crystallized and studied by X-ray analysis at 2.3 and 2.8 angstroms resolutions respectively. The three-dimensional structures showed that the mutations gave rise to local conformational changes only when compared with that of chymosin B. Kinetic analysis of the A115T mutant with a six residue synthetic peptide revealed a reduction in Km with respect to the wild type, possibly caused by the small local changes in the vicinity of S1 and S3. Although, kinetic analyses of the G243D mutant using the short substrate showed reduced catalytic activity, use of a 15 residue substrate based on residues 98-112 of kappa-casein, the natural substrate, revealed an increase in the kcat compared with chymosin B, probably a consequence of the charge introduced that may interact with the substrate between P4 and P8.
Williams
M G
MG
Department of Crystallography, Birkbeck College, University of London, UK.
Wilsher
J
J
Nugent
P
P
Mills
A
A
Dhanaraj
V
V
Fabry
M
M
Sedlácek
J
J
Uusitalo
J M
JM
Penttila
M E
ME
Pitts
J E
JE
Blundell
T L
TL
D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000378:metabolism; D002417:Cattle; D012085:Chymosin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D014242:Trichoderma / Q000201:enzymology
D002364:Caseins; D012085:Chymosin